I. CELLULAR ADHESION
In order to properly defend a host against foreign invaders such as bacteria or viruses, leukocytes must be able to migrate from the circulation to sites of infection and inflammation. Leukocytes must also be able to attach to antigen-presenting cells so that a normal specific immune response can occur, and finally, they must attach to appropriate target cells so that lysis of virally-infected or tumor cells can occur. Each of these migration processes requires that leukocytes have the ability to adhere to other cells, especially endothelial cells. An excellent review of the properties and characteristics of leukocytes is provided by Eisen, H. W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, Pa. (1980), pp. 290-295 and 381-418).
Three classes of molecules have been found to have a role in mediating cellular adhesion: molecules of the integrin family, molecules of the super-immunoglobulin family, and molecules of the selectin family.
A. THE ADHESION MOLECULES OF THE INTEGRIN FAMILY
The receptor molecules of the integrin family that are involved in cellular adhesion have been termed the "CD11/CD18 family of receptor molecules." These molecules were originally identified using hybridoma technology (Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981); Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979); Springer, T. et al., Fed. Proc. 44:2660-2663 (1985)).
The receptor molecules of the CD11/CD18 family are heterodimers containing an .alpha. subunit (CD11) and a .beta. subunit (CD18) (Sanchez-Madrid, F. et al., J. Exper. Med, 158:1785-1803 (1983); Keizer, G. D. et al., Eur, J. Immunol. 15:1142-1147 (1985)). The .beta. chain of the three molecules is identical. Although the .alpha. chains of the heterodimers differ, close analysis has revealed that there are substantial similarities between them. Reviews of the similarities between the .alpha. subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al., (J. Exper. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983)).
The three different .alpha. subunits have been termed: CD11a (equivalently referred to as the LFA-1 .alpha. subunit), CD11b (equivalently referred to as the Mac-1 .alpha. subunit) and CD11c (equivalently referred to as the p150,95 .alpha. subunit) (E. Ruoslahti et al., Science 238:491 (1987); D. C. Anderson et al., Ann. Rev. Med. 38:175 (1987)).
The CD18 molecules were found to have a molecular weight of 95 kd whereas the molecular weights of the .alpha. chains were found to vary from 150 kd to 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)).
The CD11a/CD18 heterodimer is found on most lymphocytes (Springer, T. A., et al. Immunol. Rev. 68:111-135 (1982); E. Ruoslahti et al., Science 238:491 (1987); D. C. Anderson et al., Ann. Rev. Med. 38:175 (1987)). The CD11b/CD18 and CD11c/CD18 heterodimers are found on macrophages, granulocytes and large granular lymphocytes (Ruoslahti et al., Science 238:491 (1987); D. C. Anderson et al., Ann. Rev. Med. 38:175 (1987)). These three molecules play a role in cellular adhesion (Keizer, G. et al., Eur. J. Immunol. 15:1142-1147 (1985)).
The importance of the CD11a/CD18 complex and its cellular ligands in host defense has been illuminated by identification of an autosomal recessive trait (designated "LAD" Syndrome for Leukocyte Adhesion Deficiency Syndrome) characterized by recurrent, severe bacterial infections in which affected individuals are unable to synthesize normal CD18 molecules (E. Ruoslahti et al., Science 238:491 (1987); D. C. Anderson et al., Ann, Rev. Med. 38:175 (1987); Anderson, D. C., et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)). Leukocytes from these patients displayed in vitro defects similar to normal counterparts whose LFA-1 family of molecules had been antagonized by antibodies. Furthermore, these individuals were unable to mount a normal immune response due to an inability of their cells to adhere to cellular substrates (Anderson, D. C., et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)). These data show that immune reactions are mitigated when leukocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the LFA-1 family. Leukocytes from such individuals are unresponsive to stimuli which induce leukocytes to adhere to and move across vascular endothelial cells (Smith, C. W. et al., J. Clin. Invest. 82:1746 (1988)).
The CD11/CD18 complex is also involved in other cell-cell interactions involved in host defence against infection, including binding and phagocytosis of iC3b-opsonized particles, a property of CD11b/CD18 on granulocytes and monocytoid cells, and Mg.sup.2+ -dependent adhesion and killing of target cells by T cells and NK cells, a property of the CD11a/CD18 heteroduplex (E. Ruoslahti et al., Science 238:491 (1987); D. C. Anderson et al., Ann. Rev. Med. 38:175 (1987)).
B. THE ADHESION MOLECULES OF THE SUPER-IMMUNOGLOBULIN FAMILY
The natural binding ligand for the CD11/CD18 receptor molecules is Intercellular Adhesion Molecule-1 ("ICAM-1" or CD54) (Rothlein et al., J. Immunol. 137:1270 (1986)), European Patent Application Publication No. 289,949, Simmons, D. et al., Nature 331:624-627 (1988); Staunton, D. E. et al., Cell 52:925-933 (1988); which references are incorporated herein by reference).
ICAM-1 is a member of the super-immunoglobulin family of molecules. Members of this superfamily are characterized by the presence of one or more Ig homology regions, each consisting of a disulfide-bridged loop that has a number of anti-parallel .beta.-pleated strands arranged in two sheets. Three types of homology regions have been defined, each with a typical length and having a consensus sequence of amino acid residues located between the cysteines of the disulfide bond. (Williams, A. F. et al., Ann. Rev. Immunol. 6:381-405 (1988); Hunkapillar, T. et al., Adv. Immunol. 44:1-63 (1989)).
ICAM-1 is a cell surface glycoprotein of 97-114 kd. ICAM-1 has 5 Ig-like domains. Its structure is closely related to those of the neural cell adhesion molecule (NCAM) and the myelin-associated glycoprotein (MAG) (Simmons, D. et al., Nature 331:624-627 (1988); Staunton, D. E. et al., Cell 52.:925-933 (1988); Staunton, D. E. et al., Cell 61243-254 (1990), herein incorporated by reference).
ICAM-1 is inducible on fibroblasts and endothelial cells in vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor in a time frame consistent with the infiltration of lymphocytes into inflammatory lesions in vivo (Dustin, M. L., et. al., J. Immunol 137:245-254, (1986); Pober, J. S., et al., J. Immunol 137:1893-1896, (1986)). ICAM-1 is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, other epithelial cells, and fibroblasts and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center B-cells and dendritic cells in tonsils, lymph nodes and Peyer's patches (Dustin, M. L., et. al., J. Immunol 137:245-254, (1986)). ICAM-1 is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen planus, exanthema, urticaria and bullous diseases.
Thus, ICAM-1 is preferentially expressed at sites of inflammation, and is not generally expressed by quiescent cells. It functions as the cellular substrate to which lymphocytes can attach, so that the lymphocytes may migrate to sites of infection or inflammation.
ICAM-2 is a second LFA-1 ligand, distinct from ICAM-1 (Rothlein, R. et al., J. Immunol. 137:1270-1274 (1986); Makgoba, M. W. et al., Eur. J. Immunol. 18:637-640 (1988); Dustin, M. L. et al., J. Cell. Biol. 107:321-331 (1988); Staunton, D. M. et al., FASEB J. 3:a446 (1989)). Like ICAM-1, ICAM-2 is a member of the super-immunoglobulin family.
ICAM-2 is constitutively expressed on endothelial cells, and on certain interstitial cells. It is also present on a variety of T-and B-lymphoblastoid cell lines. ICAM-2 is the predominant LFA-1 ligand on unactivated endothelium. It has thus been reported to play a role in normal lymphocyte recirculation, and memory cell recruitment, and to have a role in the interaction of antigen-presenting cells (deFougerolles, A. R. et al., J. Exper. Med. 174:253-267 (1991)).
C. THE ADHESION MOLECULES OF THE SELECTIN FAMILY
The selectin (or LECCAM) family of adhesion molecules recognize and bind carbohydrate lectins. Three selectins have been described: L-selectin (also termed LECCAM-1, MEL-14, LAM-1, LECAM-1 or lymphocyte homing receptor); E-selectin (also known as endothelial leukocyte adhesion molecule-1 (ELAM-1), or LECCAM-2); and P-selectin (also known as CD62, platelet activation dependent granule external membrane (PADGEM), LECCAM-3, or granule membrane protein-140 (GMP-140)).
L-selectin is expressed by leukocytes, and plays a role in the homing of leukocytes to peripheral lymph nodes (Gallatin, M. W. et al., Nature 304:30-34 (1989); Lasky, L. A. et al., Cell 56:1045-1055 (1989); Siegelman, M. H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:5562-5566 (1989); Tedder, T. F. et al., J. Exper. Med. 170:123-133 (1989)). It is also expressed on granulocytes.
E-selectin is expressed on endothelial cells in response to cellular stimulation by cytokines such as TNF-.alpha. or IL-1.beta., or by bacterial endotoxin (LPS) (Bevilacqua, M. P. et al., Science 243:1160-1165 (1989); Tedder, T. F. et al., J. Exper. Med. 170:123-133 (1989); Bevilacqua, M. P. et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:9238-9242 (1987); Luscinskas, F. W. et al., J. Immunol. 143:3318-3324 (1989)). In vitro, E-selectin mediates the adhesion of neutrophils, monocytes, eosinophils, a subset of lymphocytes, and certain carcinoma cells. The binding ligand of E-selectin is SLE.sup.x (Lowe, J. B. et. al., Cell 63:475-484 (1990)). E-selectin is expressed in chronic inflammatory disease such as rheumatoid arthritis.
P-selectin is expressed on platelets and on endothelial cells, neutrophils, other myeloid cells, and a subset of T lymphocytes (Siegelman, M. H., Curr. Biol. 1:125-128 (1991); Pober, J. S. et al., Lab. Invest. 64:301-305 (1991)). P-selectin and E-selectin bind to a similar spectrum of cells in accordance with the fact that both can bind the SLe.sup.x lectin. The expression of P-selectin is induced by activators such as thrombin, histamine, and hydrogen peroxide.
The expression of E-selectin and P-selectin is reported to reflect inflammatory and hemostatic responses, respectively, to tissue injury (Geng, J. G. et al., Nature 343:757-760 (1990); Toothill, V. J. et al., J. Immunol. 145:283-291 (1990)). L-selectin has been reported to participate in the recruitment of cells to sites of inflammation (Watson, S. R. et al., Nature 349:164-167 (1991); Lewinsohn, D. M. et al., J. Immunol. 138:4313-4321 (1987)). All three selectins have been reported to be involved in the recruitment of neutrophils and other leukocytes to sites of inflammation (Arfors, D. E. et al., Blood 69:338-340 (1987); Smith, C. W. et al., J. Clin. Invest. 82:1746-1756 (1988); Anderson, D. C. et al., Ann. Rev. Med. 38:175-194 (1990); Larson, R. S. et al., Immunol. Rev. 114:181-217 (1990)).
The selectins have been reported to act by aiding the initial adhesion or "rolling" of neutrophils on activated endothelium (von Andrian, U. H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:7538-7542 (1991); Ley, K. et al., Blood 77:2553-2555 (1991); Lawrence, M. B. et al., Cell 65:859-873 (1991); Smith, C. W. et al., J. Clin. Invest. 83:2008-2017 (1989)). In contrast, molecules of the CD11/CD18 family are thought to mediate the subsequent arrest of the migrating neutrophils, once they have reached a site of inflammation (Anderson, D. C. et al., Ann. Rev. Med. 38:175-194 (1990); Larson, R. S. et al., Immunol. Rev. 114:181-217 (1990); Smith, C. W. et al., J. Clin. Invest. 83:2008-2017 (1989); Luscinskas, F. W. et al., J. Immunol. 146:1617-1625 (1989)).
Thus, in summary, the ability of leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells). This adherence has been found to require cell-cell contacts which involve specific receptor molecules present on the cell surface of the leukocytes and endothelium. These receptors enable a leukocyte to adhere to other leukocytes or to endothelial, and other non-vascular cells. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms including defective antibody responses.
Since cellular adhesion is involved in the process through which foreign tissue is identified and rejected, an understanding of this process is of significant value in the fields of inflammation, organ transplantation, tissue grafting, allergy and oncology.
D. OTHER ADHESION MOLECULES
VCAM-1 (vascular cell adhesion molecule - 1) is a cell surface receptor found on vascular cells (Hynes, R. O. Cell 48:549-554 (987); Price, G. E., Science 246:1303-1306 (1980)). Under normal physiologic conditions, VCAM-1 is either not expressed, or is minimally expressed. It is, however, rapidly induced upon stimulation with TNF-.alpha. or IL-1.beta.. VCAM-1 has been shown to bind to the VLA-4 integrin molecule that is expressed on leukocytes and other cells (Springer, T. A., Nature 346:425-434 (1990); Hemler, M. E. et al., Immunol. Rev. 114:45-65 (1990)). VLA-4 has been shown to mediate lymphocyte binding to endothelium of mucosal lymph nodes (Holtzmann, B. et al. Cell 56:37-46 (1989); (Holtzmann, B. et al. EMBO J. 8:1735-1741 (1989)), and to mediate cytotoxic T-cell activity (Hemler, M. E. et al., J. Biol. Chem. 262:11478-11485 (1987); Hemler, M. E. et al., In: Leukocyte Adhesion Molecules (Springer, T. A. et al., eds.) pp44-57, Springer-Verlag, N.Y. (1989)). The expression of VLA-4 is substantially unaffected by cytokines.
II. Production of Transgenic Animals: Microinjection Methods
The most widely used method through which transgenic animals have been produced involves injecting a DNA molecule into the male pronucleus of a fertilized egg (Brinster, R. L. et al., Cell 27:223 (1981); Costantini, F. et al., Nature 294:92 (1981); Harbers, K. et al., Nature 393:540 (1981); Wagner, E. F. et al., Proc. Natl. Acad. Sci. (U.S.A.) 78:5016 (1981); Gordon, J. W. et al., Proc. Natl. Acad. Sci. (U.S.A.) 73:1260 (1976); Stewart, T. A. et al., Science 217:1046-1048 (1982); Palmiter, R. D. et al., Science 222:809 (1983); Evans, R. M et al. (U.S. Pat. No. 4,870,009)).
The gene sequence being introduced need not be incorporated into any kind of self-replicating plasmid or virus (Jaenisch, R., Science, 240:1468-1474 (1988)). Indeed, the presence of vector DNA has been found, in many cases, to be undesirable (Hammer, R. E. et al., Science 235:53 (1987); Chada, K. et al., Nature 319:685 (1986); Kollias, G. et al., Cell 46:89 (1986); Shani, M., Molec. Cell. Biol. 6:2624 (1986); Chada, K. et al., Nature 314:377 (1985); Townes, T. et al., EMBO J. 4:1715 (1985)).
After being injected into the recipient fertilized egg, the DNA molecules are believed to recombine with one another to form extended head-to-tail concatemers. It has been proposed that such concatemers occur at sites of double-stranded DNA breaks at random sites in the egg's chromosomes, and that the concatemers are inserted and integrated into such sites (Brinster, R. L. et al., Proc. Natl. Acad. Sci. (U.S.A.) 82:4438 (1985)). Although it is, thus, possible for the injected DNA molecules to be incorporated at several sites within the chromosomes of the fertilized egg, in most instances, only a single site of insertion is observed (Jaenisch, R., Science, 240:1468-1474 (1988)).
Once the DNA molecule has been injected into the fertilized egg cell, the cell is implanted into the uterus of a recipient female, and allowed to develop into an animal. Since all of the animal's cells are derived from the implanted fertilized egg, all of the cells of the resulting animal (including the germ line cells) shall contain the introduced gene sequence. If, as occurs in about 30% of events, the first cellular division occurs before the introduced gene sequence has integrated into the cell's genome, the resulting animal will be a chimeric animal.
By breeding and inbreeding such animals, it has been possible to produce heterozygous and homozygous transgenic animals. Despite any unpredictability in the formation of such transgenic animals, the animals have generally been found to be stable, and to be capable of producing offspring which retain and express the introduced gene sequence.
Since microinjection causes the injected DNA to be incorporated into the genome of the fertilized egg through a process involving the disruption and alteration of the nucleotide sequence in the chromosome of the egg at the insertion site, it has been observed to result in the alteration, disruption, or loss of function of the endogenous egg gene in which the injected DNA is inserted. Moreover, substantial alterations (deletions, duplications, rearrangements, and translocations) of the endogenous egg sequences flanking the inserted DNA have been observed (Mahon, K. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:1165 (1988); Covarrubias, Y. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:6020 (1986); Mark, W. et al., Cold Spr. Harb. Symp. Quant. Biol. 50:453 (1985)). Indeed, lethal mutations or gross morphological abnormalities have been observed (Jaenisch, R., Science 240:1468-1474 (1988); First, N. L. et al., Amer. Meat Sci. Assn. 39th Reciprocal Meat Conf. 39:41 (1986)) ).
Significantly, it has been observed that even if the desired gene sequence of the microinjected DNA molecule is one that is naturally found in the recipient egg's genome, integration of the desired gene sequence rarely occurs at the site of the natural gene (Brinster, R. L. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7087-7091 (1989)). Moreover, introduction of the desired gene sequence does not generally alter the sequence of the originally present egg gene.
Although the site in the fertilized egg's genome into which the injected DNA ultimately integrates cannot be predetermined, it is possible to control the expression of the desired gene sequence such that, in the animal, expression of the sequence will occur in an organ or tissue specific manner (reviewed by Westphal, H., FASEB J. 3:117 (1989); Jaenisch, R., Science 240:1468-1474 (1988); Meade, H. et al. (U.S. Pat. No. 4,873,316)).
The success rate for producing transgenic animals is greatest in mice. Approximately 25% of fertilized mouse eggs into which DNA has been injected, and which have been implanted in a female, will become transgenic mice. A lower rate has been thus far achieved with rabbits, sheep, cattle, and pigs (Jaenisch, R., Science 240:1468-1474 (1988); Hammer, R. E. et al., J. Animal. Sci. 63:269 (1986); Hammer, R. E. et al., Nature 315:680 (1985); Wagner, T. E. et al., Theriogenology 21:29 (1984)). The lower rate may reflect greater familiarity with the mouse as a genetic system, or may reflect the difficulty of visualizing the male pronucleus of the fertilized eggs of many farm animals (Wagner, T. E. et al., Theriogenology 21:29 (1984)).
Thus, the production of transgenic animals by microinjection of DNA suffers from at least two major drawbacks. First, it can be accomplished only during the single-cell stage of an animal's life. Second, it requires the disruption of the natural sequence of the DNA, and thus is often mutagenic or teratogenic (Gridley, T. et al., Trends Genet. 3:162 (1987)).
III. Production of Chimeric and Transgenic Animals: Recombinant Viral and Retrovital Methods
Chimeric and transgenic animals may also be produced using recombinant viral or retroviral techniques in which the gene sequence is introduced into an animal at a multi-cell stage. In such methods, the desired gene sequence is introduced into a virus or retrovirus. Cells which are infected with the virus acquire the introduced gene sequence. If the virus or retrovirus infects every cell of the animal, then the method results in the production of a transgenic animal. If, however, the virus infects only some of the animal's cells, then a chimeric animal is produced.
The general advantage of viral or retroviral methods of producing transgenic animals over those methods which involve the microinjection of non-replicating DNA, is that it is not necessary to perform the genetic manipulations at a single cell stage. Moreover, infection is a highly efficient means for introducing the DNA into a desired cell.
Recombinant retroviral methods for producing chimeric or transgenic animals have the advantage that retroviruses integrate into a host's genome in a precise manner, resulting generally in the presence of only a single integrated retrovirus (although multiple insertions may occur). Rearrangements of the host chromosome at the site of integration are, in general, limited to minor duplications (4-6 base pairs) of host DNA at the host virus junctions (Jaenisch, R., Science 240:1468-1474 (1988); see also, Varmus, H., In: RNA Tumor Viruses (Weiss, R. et al., Eds.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 369-512 (1982)). The method is, however, as mutagenic as microinjection methods.
Chimeric animals have, for example, been produced by incorporating a desired gene sequence into a virus (such as bovine papilloma virus or polyoma) which is capable of infecting the cells of a host animal. Upon infection, the virus can be maintained in an infected cell as an extrachromosomal episome (Elbrecht, A. et al., Molec. Cell. Biol. 7:1276 (1987); Lacey, M. et al., Nature 322:609 (1986); Leopold, P. et al., Cell 51:885 (1987)). Although this method decreases the mutagenic nature of chimeric/transgenic animal formation, it does so by decreasing germ line stability, and increasing oncogenicity.
Pluripotent embryonic stem cells (referred to as "ES" cells) are cells which may be obtained from embryos until the early post-implantation stage of embryogenesis. The cells may be propagated in culture, and are able to differentiate either in vitro or in vivo upon implantation into a mouse as a tumor. ES cells have a normal karyotype (Evans, M. J. et al., Nature 291:154-156 (1981); Martin, G. R. et al., Proc. Natl. Acad. Sci. (U.S.A.) 78:7634-7638 (1981)).
Upon injection into a blastocyst of a developing embryo, ES cells will proliferate and differentiate, thus resulting in the production of a chimeric animal. ES cells are capable of colonizing both the somatic and germ-line lineages of such a chimeric animal (Robertson, E. et al., Cold Spring Harb. Conf. Cell Prolif. 10:647-663 (1983); Bradley A. et al., Nature 309:255-256 (1984); Bradley, A. et al., Curr. Top. Devel. Biol. 20:357-371 (1986); Wagner, E. F. et al., Cold Spring Harb. Symp. Quant. Biol. 50:691-700 (1985); (all of which references are incorporated herein by reference).
In this method, ES cells are cultured in vitro, and infected with a viral or retroviral vector containing the gene sequence of interest. Chimerio animals generated with retroviral vectors have been found to have germ cells which either lack the introduced gene sequence, or contain the introduced sequence but lack the capacity to produce progeny cells capable of expressing the introduced sequence (Evans, M. J. et al., Cold Spring Harb. Symp. Quant. Biol. 50:685-689 (1985); Stewart, C. L. et al., EMBO J. 4:3701-3709 (1985); Robertson, L. et al., Nature (1986); which references are incorporated herein by reference).
Because ES cells may be propagated in vitro, it is possible to manipulate such cells using the techniques of somatic cell genetics. Thus, it is possible to select ES cells which carry mutations (such as in the hprt gene (encoding hypoxanthine phosphoribosyl transferase) (Hooper, M. et al., Nature 326:292-295 (1987); Kuehn, M. R. et al., Nature 326:295-298 (1987)). Such selected cells can then be used to produce chimeric or transgenic mice which fail to express an active HPRT enzyme, and thus provide animal models for diseases (such as the Lesch-Nyhan syndrome which is characterized by an HPRT deficiency) (Doetschman, T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:8583-8587 (1988)).
As indicated above, it is possible to generate a transgenic animal from a chimeric animal (whose germ line cells contain the introduced gene sequence) by inbreeding.
The above-described methods permit one to screen for the desired genetic alteration prior to introducing the transfected ES cells into the blastocyst. One drawback of these methods, however, is the inability to control the site or nature of the integration of the vector.
IV. Production of Chimeric and Transgenic Animals: Plasmid Methods
The inherent drawbacks of the above-described methods for producing chimeric and transgenie animals have caused researchers to attempt to identify additional methods through which such animals could be produced.
Gossler, A. et al., for example, have described the use of a plasmid vector which had been modified to contain the gene for neomycin phosphotransferase (nptII gene) to transfect ES cells in culture. The presence of the nptII gene conferred resistance to the antibiotic G418 to ES cells that had been infected by the plasmid (Gossler, A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:9065-9069 (1986), which reference is incorporated herein by reference). The chimeric animals which received the plasmid and which became resistant to G418, were found to have integrated the vector into their chromosomes.
Takahashi, Y. et al. have described the use of a plasmid to produce chimeric mice cells which expressed an avian crystallin gene (Development 102:258-269 (1988), incorporated herein by reference). The avian gene was incorporated into a plasmid which contained the nptII gene. Resulting chimeric animals were found to express the avian gene.
V. Production of Chimeric and Transgenic Animals: Gene Targeting Methods
One approach to producing animals having defined and specific genetic alterations has used homologous recombination to control the site of integration of an introduced marker gene sequence in tumor cells and in fusions between diploid human fibroblast and tetraploid mouse erythroleukemia cells (Smithies, O. et al., Nature 317:230-234 (1985)).
This approach was further exploited by Thomas, K. R., and co-workers, who described a general method, known as "gene targeting," for targeting mutations to a preselected, desired gene sequence of an ES cell in order to produce a transgenic animal (Mansour, S. L. et al., Nature 336:348-352 (1988); Capecchi, M. R., Trends Genet. 5:70-76 (1989); Capecchi, M. R., Science 244:1288-1292 (1989); Capecchi, M. R. et al., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 45-52; Frohman, M. A. et al., Cell 56:145-147 (1989); all of which references are incorporated herein by reference).
It may now be feasible to deliberately alter any gene in a mouse (Capecchi, M. R., Trends Genet. 5:70-76 (1989); Frohman, M. A. et al., Cell 56:145-147 (1989)). Gene targeting involves the use of standard recombinant DNA techniques to introduce a desired mutation into a cloned DNA sequence of a chosen locus. That mutation is then transferred through homologous recombination to the genome of a pluripotent, embryo-derived stem (ES) cell. The altered stem cells are microinjected into mouse blastocysts and are incorporated into the developing mouse embryo to ultimately develop into chimeric animals. In some cases, germ line cells of the chimeric animals will be derived from the genetically altered ES cells, and the mutant genotypes can be transmitted through breeding.
Gene targeting has been used to produce chimeric and transgenic mice in which an nptII gene has been inserted into the .beta..sub.2 -microglobulin locus (Koller, B. H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:8932-8935 (1989); Zijlstra, M. et al., Nature 342:435-438 (1989); Zijlstra, M. et al., Nature 344:742-746 (1989); DeChiaba et al., Nature 345:78-80 (1990)). Similar experiments have enabled the production of chimeric and transgenic animals having a c-abl gene which has been disrupted by the insertion of an nptII gene (Schwartzberg, P. L. et al., Science 246:799-803 (1989)). The technique has been used to produce chimeric mice in which the en-2 gene has been disrupted by the insertion of an nptII gene (Joyner, A. L. et al., Nature 338:153-155 (1989)).
Gene targeting has also been used to correct an hprt deficiency in an hprt.sup.- ES cell line. Cells corrected of the deficiency were used to produce chimeric animals. Significantly, all of the corrected cells exhibited gross disruption of the regions flanking the hprt locus; all of the cells tested were found to contain at least one copy of the vector used to correct the deficiency, integrated at the hprt locus (Thompson, S. et al., Cell 56:313-321 (1989); Koller, B. H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:8927-8931 (1989)).
In order to utilize the "gene targeting" method, the gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in the insertion of a marker gene (i.e. the nptII gene) into a translated region of a particular gene of interest. Thus, use of the gene targeting method results in the gross destruction of the gene of interest.
Recently, chimeric mice carrying the homeobox hox 1.1 allele have been produced using a modification of the gene targeting method (Zimmer, A. et al., Nature 338:150-154 (1989). In this modification, the integration of vector sequences was avoided by microinjecting ES cells with linear DNA containing only a portion of the hox 1.1 allele, without any accompanying vector sequences. The DNA was found to cause the gene conversion of the cellular hox allele. Selection was not used to facilitate the recovery of the "converted" ES cells, which were identified using the polymerase chain reaction ("PCR"). Approximately 50% of cells which had been clonally purified from "converted" cells were found to contain the introduced hox 1.1 allele, suggesting to Zimmer, A. et al. either chromosomal instability or contamination of sample. None of the chimeric mice were found to be able to transmit the "converted" gene to their progeny (Zimmer, A. et al., In: current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 53-58).
Significantly, the use of gene targeting to alter a gene of a cell results in the formation of a gross alteration in the sequence of that gene. The efficiency of gene targeting depends upon a number of variables, and is different from construct to construct. For example, in the CD18 gene constructs used herein, such efficiency was approximately 1/300.
VI. Introduction of Gene Sequences into Somatic Cells
DNA has been introduced into somatic cells to produce variant (chimeric) cell lines. hprt-deficient Chinese hamster ovary (CHO) cells have been transformed with the CHO hprt gene in order to produce a prototrophic cell line (Graf, L. H. et al., Somat. Cell Genet. 5:1031-1044 (1979)). Folger et al. examined the fate of a thymidine kinase gene (tk gene) which had been microinjected into the nuclei of cultured mammalian cells. Recipient cells were found to contain from 1 to 100 copies of the introduced gene sequence integrated as concatemers at one or a few sites in the cellular genome (Folger, K. R. et al., Molec. Cell. Biol. 2:1372-1387 (1982)). DNA-mediated transformation of an RNA polymerase II gene into Syrian hamster cells has also been reported (Ingles, C. et al., Molec. Cell. Biol. 2:666-673 (1982)).
Plasmids conferring host neomycin resistance and guanosine phosphotransferase activity have been transfected into Chinese hamster ovary cells to generate novel cell lines (Robson, C. N. et al., Mutat. Res. 163:201-208 (1986)).
VII. Conclusions
The application of the above-described technologies has the potential to produce animals which cannot be produced through classical genetics. For example, animals can be produced which suffer from human diseases (such as AIDS, diabetes, cancer, etc.), and may be valuable in elucidating therapies for such diseases. Chimeric and transgenic animals have substantial use as probes of natural gene expression.
Leder, P. et al. (U.S. Pat. No. 4,736,866) disclose the production of transgenic non-human mammals which contain cells having an exogenously added activated oncogene sequence. Although the animals are disclosed as being useful for assaying for carcinogenic materials, the precise location and structure of the added oncogene sequence in the animals is unknown, and cannot be experimentally controlled. Thus, the value of the animals as a model for oncogenesis is significantly impaired.
Recently, Donehower, L. A. et al. have described the construction of a transgenic mouse that carries a mutation in a chromosomal p53 allele (Donehower, L. A. et al., Nature 356:215-221 (1992), herein incorporated by reference). Such an animal is of great importance in studies of tumor suppression and oncogenesis.
Despite the successes of the above-described techniques, the methods have not yet led to the development of a model transgenic animal which can be used to study the conditions responsible for the LAD, or inflammatory responses, in general, and which can be used as a means for developing suitable anti-inflammatory agents and therapies. If however, such animals could be obtained, they would facilitate a better understanding of the inflammatory process; they could be used to assay for the presence of agonists or antagonists of inflammation; they could also be used to identify agents capable of suppressing or preventing cancer, atherosclerosis, transplantation rejection, and autoimmune disease. For example, if mutations which reduce the expression of CD18, CD11a, CD11b, CD11c, VLA-4, ICAM-1, ICAM-2, VCAM-1, P-selectin, E-selectin, or L-selectin, protect an animal against atherosclerosis, transplantation rejection, inflammatory processes, tumor metastasis, or other disease processes, this would be strong evidence that drugs which block the adhesion sites of these proteins would also protect against these disease processes. Thus, chimeric and transgenic animals having altered alleles of these genes would be extremely desirable. The present invention provides such animals, and the methods to produce and use them.